The long-term objective of this project is to understand how the unique structure of the mammalian cone photoreceptor synapse determines its function in vision. The opsin proteins in the outer segments of cones convert absorbed light into a voltage signal. In a necessary step for visual perception, the voltage signal spreads to the photoreceptor synaptic terminal where it gates a Ca2+ channel that controls the release of the transmitter glutamate onto postsynaptic bipolar and horizontal cells. Parallel processing in the visual system begins at the cone synapse. Each cone terminal communicates with ~14 anatomically distinct bipolar cell types at two structurally unique contacts termed invaginating and basal. Transmitter is not released at basal contacts, but instead at sites near the top of each of a cone's 20-40 invaginations. Released glutamate must then diffuse over an extracellular path of 200 ? 500 nm to reach the dendrites of basally contacting bipolar cells. Recent results suggest that a long diffusion path can introduce a threshold that eliminates the low-amplitude noise associated with random fluctuations in cone transmitter release in the dark. At the same time, the threshold permits the cone to transmit signals resulting from larger release events coordinated by a change in illumination. Using electro- and opto-physiological techniques, this proposal addresses two mechanisms that increase the threshold nonlinearity at basal contacts: First, at least one type of Off bipolar cell expresses receptors with an unusually high EC50 for glutamate (~1.5 mM); and second, basally located glutamate transporters provide saturable binding sites that can deplete cleft glutamate under dark release conditions. Specific Aim 1 addresses the mechanisms and functions of the threshold nonlinearity at the cone to cb1a bipolar cell basal synapse. Experiments will determine how transporter glutamate binding and kainate receptor properties contribute to nonlinear signal transmission during a light stimulus. Specific Aim 2 focuses on the ?nano-scopic? spatial localization of the proteins that shape transmission at the cone synapse. This aim uses a newly developed ?thick slab? superresolution imaging technique to relate the cone synapse nanostructure to its response properties. Information about the properties of cone transmitter release, glutamate transporters, and postsynaptic receptors will be combined with localization information obtained from superresolution microscopy to create a functional model of the basal synapse. In addition to responding to membrane voltage, it is becoming increasingly clear that Ca2+ channels in the cone terminal integrate modulatory inputs from other sites in the retina including from horizontal cells. Blue or short wavelength-sensitive (S-) cones are unique among the photoreceptor types in expressing S-opsin both in the outer segment and at the synaptic terminal. Recent experiments show that when activated by light, terminal S-opsin enhances the Ca2+ current which in turn augments transmitter release. Specific Aim 3 uses electrophysiological techniques to address both the mechanisms of the S-opsin mediated Ca2+ current increase and the role of this enhancement in visual function.